The present invention relates to a cloned gene which encodes an enzyme, the purified enzyme, and the applications and products resulting from the use of the gene and enzyme.
Polysaccharides, sugars, and lipids are the primary constituents of many food and industrial products. These products are central to human and animal nutrition and therefore have significant commercial value. Crop plants are a primary source of these compounds. Numerous crop improvement efforts around the world are focused on altering the amounts and ratios of these compounds in various species.
Carbohydrates are a major form of energy storage in plants and animals. Long chain storage carbohydrates can take many forms, but most commonly are polymers of glucose molecules; these polymers are referred to as glucans. The glucose molecules in these polysaccharides can be joined together through a variety of different linkages. The storage polysaccharides starch and glycogen are .alpha.-1,4, linked glucans. Starch is the primary storage glucan in higher plants, and starch reserves in seeds comprise a major source of carbohydrate for human and animal nutrition and for the production of certain commodity chemicals such as ethanol. Glycogen is a common energy storage compound for animals and many microbes. .beta.-1,3 linked glucans are present as storage carbohydrates in numerous algal taxa, including laminarin (Phaeophyceae), chrysolaminarin (Bacillariophyceae and Chrysophyceae), and paramylon (Euglenophyceae). The .beta.-1,4 linked glucans include the structural polymer cellulose, which is one of the most abundant biological polymers on earth.
The biosynthetic pathways of all of these glucans share a common mechanistic theme: the basic building block (i.e, the substrate for chain elongation) is an activated glucose molecule. Activation is achieved by conjugation of glucose to a nucleoside diphosphate (NDP) such as uridine diphosphate (UDP) or adenosine diphosphate (ADP). Actual synthesis of the polymer is mediated by synthase enzymes (e.g., starch synthase), which utilize NDP-glucose molecules as substrates. The general reaction catalyzed by glucan synthases is shown below: EQU NDP-glucose+(glucose).sub.n .fwdarw.(glucose).sub.n+1 +NDP
Sucrose is the principal carbon transport molecule in higher plants and is an important food ingredient. The biosynthesis of sucrose also utilizes a nucleoside diphosphate-activated glucose molecule, generally UDP-glucose. The biosynthesis of many other disaccharides and complex sugars also uses NDP-glucose as a substrate.
Specific enzymes are responsible for formation of the NDP-glucose molecules that are used for glucan and complex sugar biosynthesis. UDP-glucose is formed through the action of the enzyme UDP-glucose pyrophosphorylase (E.C. 2.7.7.9; also known as glucose-1-phosphate uridylyltransferase, and hereinafter referred to as UGPase). The reaction catalyzed by UGPase is as follows: EQU glucose-1-phosphate+UTP.fwdarw.UDP-glucose+PP.sub.i
where UTP is uridine triphosphate and PP.sub.i is pyrophosphate. The subsequent hydrolysis of pyrophosphate to orthophosphate is responsible for driving the reaction toward the formation of UDP-glucose. UGPase genes have been cloned from disparate sources, including potato (Katsube et al., J. Biochem. 108: 321-326 (1990)), human (Peng and Chang, FEBS Lett. 329:153-158 (1993)), and yeast (Purnelle et al., Yeast 8:977-986 (1992)). The UGPase enzyme has also been purified from various sources, including barley (Elling and Kula, J. Biotechnol. 34:157-163 (1994)), rice (Kimura et al., Plant Physiol. Biochem. 30:683-693 (1992)), and potato (Nakano et al., J. Biochem. 106:528-532 (1989)).
The substrate for UGPase, glucose-1-phosphate, is synthesized from glucose-6-phosphate through the action of the enzyme phosphoglucomutase (E.C. 5.4.2.2; hereinafter referred to as PGMase), as shown below: EQU glucose-6-phosphate.revreaction.glucose-1-phosphate
The PGMase enzyme has been purified from many sources, including pea (Galloway and Dugger, Physiol. Plant. 92:479-486 (1994)), human (Fazi et al., Prep. Biochem. 20:219-240 (1990)), and Lactobacillus (Marechal et al., Arch. Biochem. Biophys. 228:592-599 (1984)). The PGMase gene has also been cloned from a number of sources, including human (Putt et al., Biochem. J. 296:417-422 (1993)), yeast (GenBank accession no. X72016), and E. coli (GenBank accession no. U08369).
The present invention concerns a novel carbohydrate biosynthesis gene isolated from a microalga. Microalgae are defined as unicellular, eukaryotic algae. Although their current biotechnological utilization is primarily for the production of high value specialty products, microalgae have very high productivity rates that could support the large-scale, commercial production of lower value carbohydrates and lipids. One of the species under consideration for such uses is the centric diatom Cyclotella cryptica. This organism grows naturally in salt water and has been shown to be highly productive in outdoor culture (Weissman and Tillett, NREL/TP-232-4147:32-56, (1989)). C. cryptica is under consideration for the production of alternative fuels because lipids can comprise up to 40-60% of the cellular dry weight when cells are grown under nutrient-limiting conditions. These lipids are similar in composition to the triacylglycerols produced by oilseed crops and can be readily converted, via transesterification with a simple alcohol, into a diesel fuel replacement.
In addition to its ability to accumulate lipids, C. cryptica produces a substantial amount of carbohydrate. Approximately 20-30% of the dry weight of C. cryptica cells consists of a .beta.-1,3 linked glucan referred to as chrysolaminarin (Roessler, J. Phycol. 23:494-498 (1987)). This glucan accumulates in all growth phases and decreases only slightly upon the induction of lipid accumulation in nutrient-deficient cells. Thus, this carbohydrate constitutes a significant sink for fixed carbon, and therefore competes for carbon substrates with the lipid biosynthetic pathway. Roessler (Roessler, J. Phycol. 23:494-498 (1987)) demonstrated previously that the precursor for chrysolaminarin biosynthesis in C. cryptica is UDP-glucose, and that UGPase enzyme activity was present in extracts of C. cryptica cells. The UDP-glucose produced by UGPase is a substrate for the enzyme chrysolaminarin synthase, which adds glucose units successively onto the growing carbohydrate polymer. In contrast, PGMase in C. cryptica has not been characterized.
The instant invention is directed to the isolation of a gene from C. cryptica that encodes a multifunctional enzyme that has both UGPase and PGMase activities. This is the first report of the isolation of a gene encoding either of these enzymes from an alga. The fact that UGPase and PGMase domains are both present on a single polypeptide chain could not have been anticipated; these activities have never before been reported to exist together on a single protein. Uttaro and Ugalde (Uttaro and Ugalde, Gene 150:117-122 (1994)) reported a chromosomal cluster in the bacterium Agrobacterium tumefaciens that encodes ADP-glucose pyrophosphorylase, glycogen synthase, and PGMase; however, the three activities are encoded by three separate open reading frames, and are consequently found on three separate proteins. The presence of UGPase and PGMase on a single polypeptide chain could have significant advantages both in terms of more favorable reaction kinetics and because a single gene can be inserted into an organism via genetic engineering to affect two enzymatic functions simultaneously. Artificial polypeptide fusions have been shown to have kinetic advantages in other systems (for example, Tamada et al., Bioconjugate Chem. 5:660-665 (1994)). A naturally-occurring fusion may exhibit even greater kinetic advantages than man-made fusions, in that evolutionary selective pressure can result in functionally superior enzymes.